1. Field of the Invention
The invention in the fields of biochemistry, neuroscience and medicine, relates to compositions and methods based on hybrid polypeptides including the neuregulin heparin binding domain (N-BBD) and erbB4 extracellular domain to target polypeptides to cell surfaces and to extracellular matrix rich in heparan sulfate proteoglycans (HSPGs) for the treatment of various diseases, particularly cancer.
2. Description of the Background Art
To carry out their diverse physiological functions, cells must adhere in a specific manner to cellular and extracellular components of their environment. Cells' ability to recognize multiple environmental cues and undergo specific adhesive reactions is critical to complex cellular functions. Recognition and adhesion are mediated by cell adhesion molecules (“CAMs”) which bind to macromolecules expressed on neighboring cells or in the extracellular matrix (“ECM”).
Three motifs present in adhesion molecules for which 3D structure is known are immunoglobulin (Ig) superfamily domains, fibronectin type III (Fn-III) domains and cadherin the domains. In the nervous system, Ig superfamily members mediate Ca-independent homophilic and heterophilic binding. The extracellular regions of these molecules include one or more domains (“extracellular domains” or “ECDs”) with sequence similarity to variable (V) or constant (C) domains of Ig's (Williams, A F et al., Annu. Rev. Immunol. 6:381-405 (1988). Yoshihara, Y et al., Neurosci. Res. 10:83-105 (1991). Many Ig superfamily molecules consist of tandem Ig-like domains linked in series with multiple copies of a second building block domain (e.g., an Fn-III repeat). Because two molecules that share detectable sequence similarity adopt the same folding topology, investigators have used structures of molecules discovered in studies of the immune system as “first order” models for the structures of Ig domains in neural CAMs. Ig-like domains, and their topology are reviewed in Vaughn D E et al., Neuron 16:261-273 (1998). Ig V domains, the prototype of the V-like domains of CAMs, are folded similarly; they are found in antibody VH and VL domains and the N-terminal domains of the T cell receptor (TCR) α and β chains, the T cell surface molecule CD4 (first and third domains) and CD8, the N-terminal domains of the “immune system CAM” CD2, vascular cell adhesion molecule-1 (VCAM-1) and telokin, and the C-terminal domain of the myosin light chain kinase. Ig C1 domains consist of seven β strands arranged into two antiparallel sheets. The two sheets are connected by a disulfide bond between strands “B” and “F.” In an antibody, constant domains are found in the Fc regions and the C-terminal domains of the Fab Ig fragment. Constant-like or C1 set domains, are also found in the membrane proximal domains of MHC molecules and TCRs. The C2 and C1 folding topologies are similar. C2 domains are present in three Ig superfamily members: CD2, the second domain of VCAM-1 and the second and fourth domains of CD4. The heparin binding domain (HBD) of neuregulin, a focus of the present invention, is an Ig-C2 domain.
Important means of intercellular communication are growth and differentiation factors that are released from one cell and bind to and activate membrane receptors on a nearby cell, which ultimately changes its properties through changes in gene expression. Many released polypeptide factors have additional binding interactions with heparan-sulfate proteoglycans (HSPGs) situated in the ECM between cells. This dual-binding interaction may serve to concentrate these factors at sites where they are needed, to protect them from proteolysis, and to modulate their interactions with their receptors (Schlessinger, J et al. (1995) Cell 83:357-360). The understanding of how these extracellular interactions modulate the intracellular events that ultimately change a cell's properties are still evolving.
The neuregulins (NRGs) are a family of heparin-binding growth and differentiation factors with multiple functions in (a) growth and development of the nervous system and heart, and (b) cancer (Fischbach, G D et al. (1997) Annu Rev Neurosci 20:429-458). In one case, NRGs released from motor nerve endings at neuromuscular synapses activate members of the epidermal growth factor (EGF) family of tyrosine kinase receptors erbB2, erbB3 and erbB4 in the postsynaptic muscle membrane (Loeb, J A et al. (1999) Development 126:781-791; Goodearl, A D et al. (1995) J Cell Biol 130: 423-1434). As discussed herein, NRGs are potent mitogens that are often released from certain tumor cells thus acting in an autocrine manner to activate the same family of receptors on the same tumor cells, resulting in enhanced proliferation and metastatic activity (Li Q et al. (2004) Cancer Res 64:7078-85).
A common feature of all NRGs is the epidermal growth factor-like (EGF-like) domain. This domain, even when expressed by itself, is sufficient for receptor binding and activation of homo- and heterodimers of erbB2, erbB3, and erbB4 receptors which are highly concentrated, for example, at the neuromuscular synapses in the postsynaptic muscle membrane (Altiok, N. et al. (1995) EMBO J 14: 4258-4266). Rapid autophosphorylation of the receptors' Tyr residues initiates a signaling cascade that translates the initial binding event into the induction of AChR genes (Corfas, G. et al. (1993) Proc. Natl Acad Sci USA 90, 1624-1628). This signaling cascade involves a number of signaling pathways including both the mitogen-activated protein (MAP) kinase pathway and phosphatidylinositol 3-kinase (PI3K) pathways (Si, J. et al. (1996) J Biol Chem 271:19752-19759; Tansey, M G et al. (1996) J Cell Biol 134, 465-476; Altiok, N et al. (1997) EMBO J 16:717-725).
Most spliced forms of NRG also have an immunoglobulin-like (IG-like) domain N-terminal to the EGF-like domain (FIG. 1). Because this domain is a heparin-binding domain (“HBD”) it is referred to herein as the neuregulin HBD (or “N-HBD”). The terms “IG-like domain” (from NRG) and “N-HBD” are used interchangeably here.
The present inventor and others have shown that the N-HBD interacts with HSPGs and may lead to the deposition of NRGs in the ECM of neuromuscular synapses and within the central nervous system (CNS) (Loeb et al., supra; Loeb, J A et al. (1995) J Cell Biol 130, 127-135; Meier, T. et al. (1998) J Cell Biol 141, 715-726). In the developing nervous system, HSPGs may “direct” the accumulation of NRG forms that include the N-HBD to the basal lamina of developing neuromuscular synapses at key stages of development (Loeb et al., 1999, supra).
One distinguishing feature of NRG is the presence of distinct domains, separated by a glycosylated spacer region, for heparan sulfate binding and for receptor binding. Recognition of this fact led the present inventor to determine the direct effects of HSPG binding on receptor activation and gene activation.
Rio, C et al., Neuron 19:39-50 (1997) described a 27 amino acid peptide of chick NRG that corresponded to the HBD, which was produced only for use as an immunogen to produce an antiserum in rabbits. Loeb, J A et al., 1995, supra, speculated that immobilization of NRGs to the ECM might involve their Ig-like domains binding to HSPGs. This speculation was based indirectly on observations that heparin inhibited receptor tyrosine phosphorylation induced by recombinant NRGs.
Since NRGs bind to heparin, T. Meier et al. (J Cell Biol, 1998, 141:715-726) examined whether recombinant HRG (=NRG) cloned from a human cDNA library bound directly to the recombinant HSPG chick agrin by the negatively charged glycosaminoglycan (GAG) side chains as proposed by Loeb et al., supra. Indeed, the Ig-like domain mediated binding to these GAG chains. The Ig-like domain, but not the EGF-like domain, bound to agrin.
While there have been numerous disclosures of Ig-C regions or various parts of Ig molecules fused to other proteins for various purposes, these regions primarily are derived from true Ig molecules. The N-HBD described here has less than 40% homology or sequence similarity to these true Ig domains so as to be distinct structurally and functionally from those in the prior art. Examples of such disclosures include the following. U.S. Pat. Nos. 5,116,964 and 5,428,130 describe a ligand, completely distinct from the NRG-HBD neuregulin IG domain described herein, that was said to target active peptides to cell surfaces. However, such targeting is not directed to, nor specific for, heparan sulfates at the cell surfaces. U.S. Pat. No. 5,565,335 describes an “immunoadhesion” comprising a fusion protein in which a polypeptide making up the adhesion variable (V) region is fused at its C-terminus to the N-terminus of an Ig C region polypeptide.
U.S. Pat. No. 6,018,026 and U.S. Pat. No. 5,155,027 describe biologically active polypeptides (and their coding DNA), and, specifically, dimerized fusion products comprising a first and a second polypeptide chain, each of which comprises a non-Ig polypeptide and requires dimerization for biological activity, joined to a heterologous “dimerizing” protein. Also described is a polypeptide chain of the non-Ig polypeptide dimer, joined to at least one Ig H chain C region domain (any of CH1-CH4). The expressed, dimerized fusion polypeptide exhibits biological activity characteristic of the non-Ig polypeptide dimer.
U.S. Pat. No. 5,541,087 describes DNA encoding a fusion protein comprising a sequence encoding an Ig Fc region lacking at least the CH1 domain, and a target protein sequence. U.S. Pat. No. 5,869,046 discloses a method for preparing a variant “polypeptide of interest” which is an Fab or a (Fab′)2 fragment, the Ig domain (or Ig-like domain) of which comprises at least one of a CH1 or CL region. U.S. Pat. No. 6,121,022 discloses a modified polypeptide having an Ig C domain or an Ig-like C domain and an epitope that binds to a salvage receptor within the Ig- or Ig-like C domain. This epitope, absent from the unmodified polypeptide, is taken from two loops of the CH2 domain of an Ig Fc region. The Ig-like domains described in these documents are clearly distinct from the N-HBD of the present invention.
U.S. Pat. No. 6,121,415 describes a family of polypeptides, collectively called neuregulins (NRG1) that appear to result from alternate splicing of a single gene mapped to the short arm of human chromosome 8 (Orr-Urtreger et al. (1993) Proc Natl Acad Sci USA 90:1867-71). The NRG3s (murine and human) were disclosed as being about 713 and 720 amino acids in length, respectively, and to comprise an EGF-like domain, an N-terminal hydrophobic segment, the Ser/Thr-rich portion, a predicted transmembrane domain, and a predicted intracellular domain.
Holmes et al. (Science (1992) 256:1205-10; WO 92/20798; and U.S. Pat. No. 5,367,060) described isolation and cloning of a family of polypeptide activators for the HER2 receptor which they called heregulin-α (HRG-α), heregulin-β1 (HRG-β1), heregulin-β2 (HRG-β2), heregulin-β2-like (HRG-β2-like), and heregulin-β3 (HRG-β3). These documents describe (1) the ability of the purified HRG (=NRG) polypeptides to activate tyrosine phosphorylation of the HER2 receptor in MCF7 breast tumor cells and (2) the mitogenic activity of the HRG polypeptides on tumor cells expressing high levels of the HER2 receptor. Like other EGF family growth factors, soluble HRG polypeptides appear to be derived from a membrane bound precursor (pro-HRG) which is proteolytically processed to release the 45 kDa soluble form. Although substantially identical in the first 213 amino acid residues, the HRGs are classified into two major types, α and β, based on two variant EGF-like domains which differ in their C-termini. Based on an amino acid sequence comparison between the first and sixth Cys residues in the EGF-like domain, HRGs were 45% similar to heparin-binding EGF-like growth factor (HB-EGF), 35% identical to amphiregulin, 32% identical to TGF-α, and 27% identical to EGF.
Falls et al. (1993) Cell 72:801-815 described a chicken heregulin family member named “acetylcholine receptor inducing activity” (ARIA) polypeptide, that stimulated synthesis of muscle AChRs. See also WO 94/08007. ARIA is a β type HRG and lacks the entire spacer region rich in glycosylation sites between the Ig-like domain and EGF-like domain of HRGα, and HRGPβ1-β3.
Marchionni et al. (1993) Nature 362:312-318, identified several bovine-proteins named glial growth factors (GGFs) which share the Ig-like domain and EGF-like domain with the other NRG/HRG proteins described above, but which also have an N-terminal kringle domain. See also WO 92/18627; WO 94/00140; WO 94/04560; WO 94/26298; and WO 95/32724.
Ho et al. (1995) J. Biol. Chem. 270:14523-32, described another member of the HRG family called “sensory and motor neuron-derived factor” (SMDF) which has an EGF-like domain characteristic of all other HRG polypeptides but a distinct N-terminal domain. The major structural difference between SMDF and the other HRG polypeptides is the lack of an Ig-like domain and the “glyco” spacer characteristic of all the other HRG polypeptides.
Caraway et al. (1994) J Biol. Chem. 269:14303-06 subsequently demonstrated that ErbB3 is a receptor for HRG and mediates phosphorylation of intrinsic Tyr residues and of ErbB2 receptor in cells which express both receptors. HRG was the only known member of the EGF-like family that could interact with several receptors (Carraway et al. (1994) Cell 78:5-8.
A number of biological activities of the NRG/HRG proteins have been described:    (1) myotube differentiation via synthesis and concentration of neurotransmitter receptors in the postsynaptic muscle (Falls et al., supra);    (2) increased number of sodium channels in chick muscle (Corfas et al., 1993, J. Neuroscience 13:2118-25);    (3) mitogenic stimulation of subconfluent quiescent human myoblasts and their differentiation to yield more myotubes (Sklar et al., WO 94/26298; and    (4) activation of myocardial ErbB2 and ErbB4 receptors by NRG1 (Ford, B D et al., Dev Biol. (1999) 214:139-50; Carraway, K L et al., Bioessays (1996) 18:263-66.
As described herein and as discussed in the present inventor's earlier application; see also Li et al., 2001, J Biol Chem. 276:38068-75).), N-HBD targets the NRG protein to the cell surface by interacting with agrin and other HSPGs in the ECM. See also: Li Q, et al., Mol. Cell. Neurosci. 26:558-69. This interaction between NRG and HSPGs not only concentrates NRG proteins near its erbB receptors, but also keeps it there for a sufficiently long time to induce biological activity (in this case, AChR gene expression). Results presented herein, supported by other reports demonstrating the mitogenic effects of NRG in breast and ovarian cancer (Aguilar, Z et al., 1999, Oncogene. 18:6050-62. Gilmour, L M et al., 2002, Clin Canc Res. 8:3933-42) indicate that blocking NRG activity is an important new method to treat these forms of cancers. For example, a naturally-occurring secreted form of human p85 erbB3 receptor negatively regulates NRG-induced breast cancer cell growth (Lee, H et al., 2001, Cancer Res. 61:4467-73). A 120 kDa fusion polypeptide named IgB4 is an NRG antagonist that includes the ECD of the erbB4 receptor fused in frame to the Fc portion of human IgG1 (hinge, CH2 and CH3 domains) (Chen, X, 1996, J Biol Chem 271:7620-9). IgB4 is dimeric because of the interchain disulfide bonds between the two Ig γ chains. Both the soluble erbB3 and the IgB4 antagonists worked as dominant-negative NRG receptors by competing with endogenous erbB receptors for NRG binding. While both dominant-negative receptors were reported to block NRG constructs that comprise the EGF-like domain alone, the present inventor and others have been unable to efficiently inhibit the activity of the Ig-EGF form of NRG (measured as erbB phosphorylation). See, for example, Li Q et al. (2004) Cancer Res 64:7078-85). According to the present invention, this failure is explained by the fact that Ig-EGF forms of NRG accumulate at the cell surface through HBD/HSPG interactions, attaining high concentrations near their natural erbB receptors. Under such circumstances, soluble antagonists cannot block NRG activity except at very high, practically unattainable, concentrations. One objective of the present invention was to overcome this deficiency.
Heparan Sulfate and HSPGs
Heparan sulfate (HS) is a sulfated polysaccharide found on the surface of most cells as part of proteoglycans (HSPGs) and in the ECM. The polysaccharide mediates the interactions between a number of different proteins. HS consists of alternating hexuronate and glucosamine units. The hexuronate can be either D-glucuronate (GlcA) or L-iduronate (IdoA). The amine of the glucosamine is usually acetylated (N-acetylglucosamine or GlcNAc) or sulfated (N-sulfoglucosamine or GlcNSO3), but it may also be unsubstituted. Potential sulfation sites located on the amino group or positions 2, 3, and 6 of each sugar molecule (Sugahara, K et al. (2002) IUBMB Life 54:163-175). Sometimes there are also 3-O-sulfate groups present on GlcNSO3 units. Expression of HS epitopes may be temporally and spatially controlled within different organs and tissues (Lindahl, U et al. (1998) J Biol Chem 273:24979-82; Esko, J D et al. (1998) J Clin Invest 108:169-173). Thus HSPG specificity may be encoded by the diversity generated by differences in sugar composition and sulfation pattern of the GAG chains (Gabius, H J (2000) Naturwissenschaften 87:108-121; Capila, I et al. (2002) Angew Chem Int Ed Engl 41:391-412). For example, two fibroblast growth factors, FGF1 and FGF2, require N-sulfated pentasaccharide sequences for optimal binding, but differ in requirements for O-sulfation (Kreuger, J. et al. (2001) J Biol Chem 276:30744-52). Hepatocyte growth factor, platelet-derived growth factor, and lipoprotein lipase all depend on the presence of one or more GlcN 6O-sulfate groups (Lyon, M et al. (1994) Biochem Soc Trans 22:365-370; Feyzi, E et al. (1997) J Biol Chem 272:5518-24; Parthasarathy, N et al. (1994) J Biol Chem 269:22391-96). Antithrombin III requires a 3O-sulfated GlcNS unit (Petitou, M et al. (1988) Carbohyd Res 179:163-72. Further specificity in the FGF signaling system may also result from either simultaneous or sequential interactions between HS and FGF and its receptors (Allen, B L et al. (2003) J Cell Biol 163:637-48; Guimond, S E et al. (1999) Curr Biol 9:1343-46).